JP6820232B2 - Purification of proteins by anion exchange chromatography - Google Patents

Description

The present invention is used in a two-step method of purifying a divalent cation-binding protein with an anion exchange resin material in high yield and with high purity, preferably in a divalent cation-binding protein that can be obtained by the method, particularly in FIX, and in the method. With respect to a kit containing means for carrying out.

Since the development of the recombination technique, in host cells, for example, transfecting the cells with DNA encoding the protein and allowing the recombinant cells to grow under favorable conditions for expressing the protein. Many mammalian proteins were produced in. Proteins secreted by the cells into the cell culture, or proteins remaining inside the cells, can be separated from the medium and other constituents using chromatography techniques such as ion exchange chromatography, affinity chromatography and the like. Furthermore, purity is particularly important for use in medicine. However, at the same time, the biological activity of the protein must be retained even after the protein of interest has been sufficiently purified.

Approximately 30 years ago, the concept of eluting calcium-binding proteins from anion exchange resins with divalent cations was first reported. Although successful isolation of bovine Factor VII from bovine plasma, purification of human Factor VII remains problematic, i.e. the substance produced is not very pure or is not a detectable protein. It was only obtained in small amounts as characterized as activity. Researchers in the field have adsorbed the protein on a divalent cation, barium citrate, and then separated the protein by anion exchange chromatography to obtain a sufficient amount of human Factor VII from human plasma (approximately). It was successfully isolated (in a yield of 30%). Furthermore, ordinary ion exchange resins such as anion exchange resins are eluted with divalent cations such as calcium ion (Ca ²⁺ ), magnesium ion (Mg ²⁺ ), barium ion (Ba ²⁺ ) and strontium ion (Sr ²⁺ ). A method of recovering and purifying a vitamin K-dependent protein from a cell medium that produces a vitamin K-dependent protein having various specific activities by using a lysate was also available.

Moreover, a solution containing Factor IX (FIX) is applied to the anion exchange resin and the anion exchange resin is washed with a lower conductive solution than required to elute the FIX from the resin. A purification method using a solution of FIX, which comprises a step of eluting FIX from the anion exchange resin with a first eluent containing a divalent cation to form a first eluate, was available. The first eluate is then applied to heparin or heparin-like resin to form a second eluate, the second eluate is applied to hydroxyaparite, and a highly conductive cleaning agent is used in the cleaning step. Form a third eluate.

Factor IX (FIX) is a vitamin K-dependent serine protease of the blood coagulation system belonging to the peptidase family S1. The FIX is inactive unless it is activated by Factor VIIa or Factor VIIa. Calcium and membrane phospholipids are required for its activation. FIX deficiency causes a recessive hereditary hemorrhagic disorder, hemophilia B, which can be treated by administering a post-translationally modified FIX, a phosphorylated and sulfated FIX. FIX can be further converted to activated FIX, i.e. FIXa. Since FIXa can adversely affect a given composition of FIX (eg, by enhancing its thrombus-forming ability as described in the literature), FIX products should prioritize low content of FIXa. is there.

Furthermore, Factor VII (FVII) is a vitamin K-dependent serine protease that plays a prominent role in the blood coagulation cascade, in which it initiates the blood coagulation process with tissue factor (TF). When blood vessels are damaged, TF is exposed to blood and circulating FVII. Once bound to TF, FVII is activated into FVIIa (by thrombin), Factor Xa, Factor IXa, Factor VIIa and the FVIIa-TF complex (its substrates are FX and TIX). Furthermore, anexin V is a cellular protein belonging to the anexin group having the ability to bind to phosphatidylserine in a calcium-dependent manner to form a membrane-bound two-dimensional crystal lattice. It may play a role in blood coagulation, apoptosis, phagocytosis and the formation of cell membrane-derived microparticles.

Thus, the underlying challenge of the present invention is to provide an improved method for purifying divalent cation-binding proteins in high yield and high purity. The above-mentioned solutions to the technical problems are achieved by embodiments characterized in the claims.

The present invention is a method for purifying a divalent cation-binding protein in a preferred embodiment (item 1).
(A) The divalent cation binding protein in the loading buffer in which the divalent cation is absent or is present at a low concentration is loaded on the first anion exchange resin material, followed by an optional 1 to 3 washing steps. ;
(B) The divalent cation-binding protein is eluted with an eluent containing a divalent cation and a counter anion to form an eluate containing the divalent cation-binding protein;
(C) Dilute the pool of resulting eluate to increase the concentration of divalent cations ((1) conductivity may be reduced as appropriate);
(D) The method comprises loading the eluate obtained in step (c) onto a second anion exchange resin material; and (e) collecting a flow-through containing a divalent cation binding protein. ..

Therefore, it is preferable that the step (a) as described above is carried out without containing a free divalent cation. In this context, "free" divalent cation means a divalent cation that is not complexed. That is, for example, if about 1 mM Ca ⁺⁺ is present in complex with EDTA, it is considered that the (free) divalent cation is not contained.

If the conductivity has not yet been reduced in the last optional cleaning step, then only the optional step C (1) to reduce the conductivity is required; as described in detail below, the conductivity is reduced. The lowering is preferably carried out in the cleaning step 3.

Item 2 After the loading step, one or more wash steps (1), (2) and / or (3) are performed with wash buffers (1), (2) and / or (3). The method of item 1, wherein the valent cation is absent but is performed in the presence of a counter anion.

Preferred counter anions are: chloride (most preferred), acetate, phosphate, sulfate, carbonate, but not limited to these.

Item 3 At least one of the loading buffer and / or the washing buffer has a pH that is at least 0.5 lower in pH units than the pH of the eluent of step (b), preferably of the loading buffer and / or washing step (2). The method of item 1 or 2, wherein any of the buffers has a pH at least 0.5 lower in pH units than the pH of the eluent of step (b).

Item 4 The eluate of step (b) has higher conductivity than the loading buffer of step (a) and any washing buffer, and the eluate replenished in step (c) is step (b). ), The method according to any one or more of items 1 to 3, which has a conductivity lower than that of the eluent.

In a preferred embodiment, the elution buffer has a conductivity of 16-25 mS / cm (RT), preferably 19-20 mS / cm (TR). When any cleaning step 2 is performed (“acidic cleaning”), the conductivity is preferably 14-19 mS / cm (RT), more preferably 16-18 mS / cm (RT), as described above in all cases. The requirements of item 4 above must be met.

Item 5 From the group consisting of at least one divalent cation in step (b) consisting of Ca ²⁺ , Be ²⁺ , Ba ²⁺ , Mg ²⁺ , Mn ²⁺ , Sr ²⁺ , Zn ²⁺ , Co ²⁺ , Ni ²⁺ , and Cu ²⁺ , or The method according to any one of items 1 to 4, which is selected from the combination.

Item 6 The primary and secondary anion exchange resin materials are diethylaminoethane (DEAE), dimethylaminoethane (DMAE), trimethylaminoethyl (TMAE), polyethyleneimine (PEI), quaternary aminoalkyl, and quaternary aminoethane, respectively. The method according to any one of items 1 to 5, which has a positively charged group independently selected from the group consisting of QAE) and quaternary ammonium (Q).

Item 7 Items 1 to 6 in which the primary and secondary anion exchange resin materials are independently selected from the group consisting of aminohexyl, benzamidine, lysine and arginine, respectively, carrying a primary amine as a ligand. The method described in any one of.

Item 8 The method according to any one of items 1 to 7, wherein the divalent cation binding protein is a calcium binding protein.

Item 9 The method according to any one of items 1 to 8, wherein the divalent cation binding protein is a vitamin K-dependent protein.

Item 10 The divalent cation-binding protein is more preferably factor IX (FIX) than the group consisting of factor VII, factor VII, factor IX, factor X, protein C, protein S, annexin and calmodulin. The method according to any one of items 1 to 9, selected from the group consisting of Factor VII (FVIIa) and Anexin V.

Any one of the above proteins can be derived either from a natural source, such as plasma, or by recombinant techniques.

Item 11 The pH of step (a) is 6.8 to 7.5, preferably 7.0 to 7.4 when any cleaning step is performed, and when no arbitrary cleaning step is performed. The method according to any one of items 1 to 10, wherein the pH is 5.5 to 6.5, preferably 5.9 to 6.1.

Item 12 Any of items 1 to 11, wherein the pH in any cleaning step (3) is 7.0 to 8.2, preferably 7.3 to 8.0, and even more preferably 7.3 to 7.5. The method described in one.

Item 13 The pH in the arbitrary cleaning step (1) is 7.3 to 7.5, and the pH in the arbitrary cleaning step (2) is preferably 5.5 to 6.5, more preferably 5.9 to 6. The method according to any one of items 2 to 12, which is .1.

Item 14 Any of items 1-13, wherein the elution buffer (ie, eluent) in step (b) contains calcium and has a pH of 7.5-8.5 (preferably 7.8-8.2). The method described in one.

Item 15 The pH of the loading buffer in the loading step (d) is higher than either the pH of the loading buffer in the loading step (a) or the pH in the washing performed after the loading (a), items 1 to 14 above. The method according to any one of, especially item 3.

In the above and also in the context of the following, "cleaning performed after loading (a)" refers herein to "acidic cleaning" or cleaning 2.

Item 16 The method according to any one of Items 1 to 15 above, particularly Item 3, wherein the conductivity in the loading step (d) is less than or equal to the conductivity in the cleaning performed after the loading (a).

Item 17 Any of the above items 1 to 16, wherein the concentration of the divalent cation in the loading buffer in step (d) is higher than the concentration of the divalent cation in the elution buffer in step (b), particularly the above. The method according to item 3.

Item 18 The step of providing low conductivity to the resulting pool of eluate is by diluting with a suitable dilution buffer, otherwise by changing the buffer composition, or by dialysis, dialysis filtration or gel filtration. The method according to any one of items 1 to 17, which is carried out.

Item 19 A divalent cation-binding protein obtained by the method according to any one of items 1 to 18 above.

Item 20 (r) FIX obtained by the method according to any one of the above items 1 to 18.

Item 21 At least 270 I. U.S. Specific activity of / mg FIX Ag, preferably 270-350 I. et al. U.S. The specific activity of / mg FIX Ag, more preferably 270-320 I. U.S. Specific activity of / mg FIX Ag, particularly preferably 280-300 I. U.S. (R) FIX according to item 20, which has a specific activity of / mg FIX Ag.

Item 22 A pharmaceutical composition comprising (r) FIX according to item 20 or 21, having a logarithmic reduction CHO HCP reduction rate of about 2.5-3.

The logarithmic reduction of 2.5-3 is the logarithm of the total reduction of CHO HCP, as also derived from Table 4 below, which Table 4 shows 229 (step 1 to step 2) and 1. A decrease in CHO HCP of 75 (from step 2 to step 3) is shown, i.e. a decrease of 1.75 x 229 = about 440 (the logarithm is 2.6).

Item 23 The (r) FIX according to item 20 or 21, which comprises a FIX Ag of less than 50 μg / mg, preferably a FIX Ag of less than 20 μg / mg, and even more preferably a CHO HCP impurity of less than 10 μg / mg. The pharmaceutical composition comprising, and / or the pharmaceutical composition according to item 22.

Item 24 The pharmaceutical composition comprising (r) FIX according to item 20 or 21, comprising CHO DNA less than 10 pg / ml, preferably less than 5 pg / ml, and even more preferably less than 1 pg / ml CHO DNA. , And / or the pharmaceutical composition according to item 22 or 23.

Item 25 (r) The FIX composition contains less than 1 mU of FIXa (activated FIX) activity / unit of FIX coagulation activity, preferably less than 0.75 mU of FIX activity / unit of FIX coagulation activity, more preferably less than 0.5 mU. FIX activity / unit of FIX coagulation activity, even more preferably less than 0.3 mU FIX activity / unit of FIX coagulation activity, and / or a pharmaceutical composition comprising (r) FIX according to item 20 or 21. The pharmaceutical composition according to any one of items 22-24.

1 mU of FIXa activity / unit of FIX coagulation activity is equal to 0.1% chromogenic activity per IU / ml FIX coagulation activity (see assay method below).

Item 26 A kit comprising means for performing the method according to any one of items 1-18.

Item 27 FIXa (activated FIX) activity of less than 1 mU / unit of FIX coagulation activity, preferably less than 0.75 mU of FIX activity / unit of FIX coagulation activity, more preferably less than 0.5 mU of FIX activity / unit of FIX coagulation. The (r) FIX composition which can be obtained by the method according to any one of items 1-18, comprising activity, more preferably less than 0.3 mU of FIX activity / unit of FIX coagulation activity.

Item 28. (R) FIX composition according to item 27, further characterized by one or more of the properties according to any one of items 20-24.

Item 29 FIXa (activated FIX) activity of less than 1 mU / unit of FIX coagulation activity, preferably less than 0.75 mU of FIX activity / unit of FIX coagulation activity, more preferably less than 0.5 mU of FIX activity / unit of FIX coagulation. (R) FIX composition comprising activity, and even more preferably less than 0.3 mU of FIX activity / unit of FIX coagulation activity.

Item 30. (R) FIX composition according to item 29, further characterized by one or more of the properties according to any one of items 20-24.

In a particularly preferred embodiment, the invention relates to the following method:

Preferably, elution (b) is carried out using an elution buffer (pH = 7.8-8.2) containing 1.8-2.2 mM calcium.

Preferably, step (c) adjusts the calcium concentration to 5-7 mM with a calcium-containing buffer to reduce the conductivity to 15-18 mS / cm (RT), resulting in a pH of 7.4-7. It is done as .8.

Thus, the present invention also relates to a kit comprising means for performing the above-mentioned method on the purified divalent cation-binding protein obtained by the above-mentioned method. Therefore, the present invention also relates to a FIX that can be obtained by the method of the present invention or is obtained by the method of the present invention, preferably a recombinant FIX. The present invention also relates to FIX or FIX compositions, preferably recombinant FIX or FIX compositions (low content of FIXa). Such FIX or FIX composition can be obtained by the method of the present invention, or obtained by the method of the present invention.

In one embodiment, the present invention is a method for purifying a divalent cation-binding protein.
(A) The divalent cation binding protein in the loading buffer in which the divalent cation is absent or is present at a low concentration is loaded on the first anion exchange resin material, followed by an optional 1 to 3 washing steps. ;
(B) The divalent cation-binding protein is eluted with an eluent containing a divalent cation and a counter anion to form an eluate containing the divalent cation-binding protein;
(C) Dilute the pool of resulting eluate to increase the concentration of divalent cations ((1) conductivity may be reduced as appropriate);
It relates to a method comprising (d) loading the eluate obtained in step (c) into a second anion exchange resin material; and (e) collecting a flow-through containing a divalent cation binding protein.

The method of the present invention uses a procedure in which two anion exchange columns are operated in a mode in which the products are bound in the first column and in a mode in which the products are not bound in the second column. The principle of the procedure is, in a preferred embodiment, a solution of a cation-binding protein (eg, rFIX) with a first anion exchange resin (on top), at a neutral or acidic pH (pH = 6.8-7.5, preferably. 7.0-7.4, eg 7.0-7.2), requires contact (ie, loading) in the presence of a chelating agent (eg, EDTA). EDTA may be selected if it is intended to further improve product yield. EDTA can improve the binding of divalent cation binding proteins, such as FIX. In a preferred embodiment, the product is washed, eg, at a pH as defined in more detail below), followed by a wash with low conductivity (this wash is just before elution) and then the product, eg calcium. Elute with the containing buffer (eg, pH = about 8.0). The pool of resulting eluate is diluted to reduce conductivity and increase calcium concentration. The diluted solution has a pH of, for example, 7.6-7.8. The pool of conditional eluents from the first anion exchange purification step is then transferred to a second anion exchange column (eg, pumped), where the cation-binding proteins, such as rFIX, are subject to the applicable conditions (especially, in particular). High concentrations of divalent cations are present and do not bind under slightly basic pH and conductive conditions of about 15-21 mS / cm (RT), whereas protein impurities bind. The resulting cation-binding protein, eg, rFIX contained in the column eluate of secondary anion exchange purification, has high affinity and high specific activity.

As used herein, the term "absence of divalent cations" means the absence of divalent cations freed in the buffer, where it is bound to a protein or confused with a chelating agent, such as EDTA. The divalent cations that are forming may be present. The term "divalent cations are present at low concentrations" means that divalent cations are present at concentrations in the range of μM, particularly up to 1000 μM, preferably up to 800 μM, and even more preferably up to 500 μM. In the present application, the term "absence of divalent cation" also means to include the above definition of "presence of divalent cation in low concentration".

As already mentioned above, protein-bound calcium, such as EDTA-bound calcium, may be acceptable in one embodiment. If calcium is bound to a protein, it is considered to be "inactive" calcium in the context of the present invention.
So the calcium does not interfere with the binding of the product. On the other hand, free calcium can interfere with product binding.

Loading of the primary anion exchange resin material with the divalent cation binding protein in the loading buffer in the absence of the divalent cation (a) can be carried out by any method known in the art. In particular, conditions suitable for loading a divalent cation binding protein into an anion exchange resin material are well known to those skilled in the art. Certain conditions of loading buffer conductivity that allow product binding depend on the individual properties of the protein and anion exchange resin material used (eg, ligand density, ligand presentation, etc.). To do. The divalent cation usually binds to the protein in the strongly acidic region (ie, the negatively charged region). When the divalent cation binds, the negative charge is shielded. However, by loading the divalent cation binding protein into the anion exchange material in the absence of the divalent cation, for example, by removing the divalent cation bound by a chelating agent (eg, EDTA), the protein becomes The surface carries a strong negatively charged compartment that allows for a strong bond to the anion exchange ligand. Furthermore, the conditions for loading a protein into an anion exchange resin material always require an equilibrium between pH and the concentration of counterions (eg, Cl ⁻ ). Counterion chemistry also affects elution behavior, for example Cl ⁻ carries a monovalent negative charge and phosphate carries a divalent negative charge at neutral pH. The latter can have high elution power even when the conductivity is low compared to Cl ⁻ .

The loading of the anion exchange column 1 (ie, step (a)) was preferably performed at a pH of 6.8-7.5, preferably 7.0-7.4, followed by 7.3 to complete the loading. Wash 1 is performed at a pH of −7.5, and wash 2 is performed under acidic conditions (pH 5.5-6.5, preferably 5.8-6.2, more preferably 5.9-6.1). To. In a further embodiment, any wash with a pH of 7.0-8.2, preferably 7.3-8.0, eg, a pH of 7.3-7.5, to prepare a column for elution. 3 (low conductivity cleaning, which is the cleaning performed immediately before elution) can be performed. Elution is carried out at a pH of 7.5-8.5, more preferably 7.8-8.2. One of the features of the present invention is that the pH of the elution buffer is at least 0.5 higher in pH units than the pH of the above-mentioned wash 2 (highly conductive wash, see the above-mentioned conductive conditions). .. This second "acidic" highly conductive cleaning has lower conductivity than elution conductivity.

In the most preferred embodiment, if any cleaning step (s) are performed, the loading step of the first anion exchange is at a neutral to slightly acidic pH, eg, 6.8-7.5. , Preferably at a pH of 7.0-7.4. Preferably, after this loading step, a first wash (pH = 7.3-7.5) is performed to complete the loading and under acidic conditions (pH = 5.5-6.5, preferably 5.8-). Perform a second wash in 6.2) and a wash 3 (pH = 7.3-7.5) to prepare a column for elution. If any cleaning step (s) are not performed, the pH of the loading step of the first anion exchange column should be 5.5-6.5, more preferably 5.9-6.1. set. In particular, one of the features of the present invention is that the pH is preferably increased by at least 0.5 in pH units before the elution step is performed. This increase can occur at all stages, for example, at the stage of the direct transition from the loading step to the elution step, even if the cleaning step is not performed in the meantime. In another embodiment, the pH increase occurs after acidic cleaning as described above. As described below, elution is preferably carried out at 7.5-8.5, more preferably at 7.8-8.2.

If the cleaning step is performed only once, i.e. if only cleaning step (1) is performed, then this cleaning step is performed without the addition of salt; as a result, the elution step is compared to the loading step. As long as the pH is finally increased by at least 0.5 in pH units, the pH is not immediately important in the washing process.

In one preferred embodiment, two cleaning steps, namely cleaning steps (1) and (2), are performed, or only stringent cleaning steps (2) (see also below) are performed. , This loading buffer has a pH in the neutral to slightly acidic range as described above, which, according to the principles of the invention, is that the second wash step (ie, wash step 2). This is possible only when the pH is as low as 5.5-6.5 or 5.9-6.1. If the loading step has already been carried out at this low pH, the washing step (2) is also preferably at this low pH. The cleaning step (2) is performed in a preferred embodiment with high conductivity as described above.

In a more preferred embodiment, if three cleaning steps, i.e., cleaning steps (1), (2) and (3), are carried out, the cleaning step (3) preferably contains no salt; the result. The above conditions mentioned in the case of the two washing steps are provided, preferably as long as the pH in the elution step is at least 0.5 greater in pH units than in the loading step and / or washing step (2). , The pH in this step is irrelevant. The cleaning (3) is preferably a low conductivity cleaning (in which case, if the resulting eluate is already low in conductivity, step c (1) does not need to be performed).

In a particularly preferred embodiment, the pH in the washing step (3) may have already been raised to the pH level in the elution step. Thus, the preferred pH of cleaning step (3) is between 7.0 and 8.2, preferably between 7.3 and 8.0, and even more preferably at 7.3-7.5. If the cleaning step (3) is performed at this particular pH condition, we will not perform the cleaning step (3) or this step will be performed at a different pH, eg, a loading step and / or a cleaning step. It has been found that the degree of impurities contained in the desired product to be eluted is further reduced compared to the situation where the pH is as low as the pH used between (1) and (2). Although not bound by the hypothesis, we believe that adjusting the pH of cleaning step (3) to the same pH as elution can avoid pH gradients during elution. The pH gradient during elution can be an interference source that allows certain impurities to elute with the product. Wash 3 described above advantageously conditions the column for elution. The high pH and low conductivity of this wash prevent impurities from elution together during the next elution.

Even more preferably, the cleaning step (3) should have very low or very close to zero conductivity, especially elution power. Such extremely low conductivity is preferably 1-15 mS / cm (RT), more preferably less than 5 mS / cm (RT).

In a preferred embodiment, the loading step (a) involves all compounds of protein, desired product, and cell culture group such as amino acids, vitamins, sugars, trace metals (KCl, NaCl, Ca, about 13 mS / cm (RT). ), Containing a solution. After the loading step, it is preferred to perform washing step 1 and thus end the loading (preferably close to neutral or neutral pH, and low conductivity. (Sex). This is preferably followed by a second cleaning step 2, which is considered to be a stringent cleaning, preferably low pH and 150-210, preferably 170-190 mM, most preferably 180 mM (which). Is carried out with NaCl, and preferred conditions with different salts are within the knowledge of those skilled in the art). In a more preferred embodiment, the washing step 3 is performed for elution. This third cleaning step is preferably carried out at low conductivity and pH as described above. This cleaning step is performed prior cleaning as described above. Has a major role of lowering the conductivity of the column and returning the pH to neutral in order to bring the column closer to the elution pH at low pH. These criteria elute with calcium and pseudo-affinity. Prepare the column to prevent impurities from elution together at the interface between the elution buffer and the wash 2 buffer.

Elution is preferably carried out in a buffer containing a divalent cation at 150-210, preferably 170-190 mM, most preferably 180 mM NaCl. In addition to the above chlorides (most preferred), other counterions, such as, but not limited to, acetates, phosphates, sulfates, carbonates, are possible, preferably 7.5-8.5. Is at a pH of 7.8-8.2, most preferably at a pH of 8.0, in any case between the pH used during loading and preferably washing during any washing. It is carried out at a pH that is at least 0.5 higher in pH units than the pH used between 2.

Furthermore, under the condition that the divalent cation binding protein binds to the anion exchange material, a loading buffer suitable for loading the divalent cation binding protein into the anion exchange material in the step (a) of the method of the present invention. Is well known in the art. As described above, if any cleaning step (s) are performed, for example, the loading buffer will have a pH of 6.8-7.5, preferably a pH of 7.0-7.4. Can have. It may contain any salt concentration suitable for binding the divalent cation binding protein to the anion exchange resin material, which can be readily determined by one of ordinary skill in the art. In a preferred embodiment, the loading buffer may contain a chelating agent, such as EDTA, preferably 0.5-10 mM EDTA, more preferably 1-5 mM EDTA, preferably about 2 mM EDTA. Chelating agents that may be separately available are also available and are well known to those of skill in the art. The loading buffer containing the divalent cation binding protein that may be applied to the anion exchange resin material by the method of the present invention may contain, for example, 20 mM MES and 2 mM EDTA. MES is an example of a buffer for pH 6; above pH 7, for example, Tris buffer can be used. In a preferred embodiment, the loading material is a cell culture supernatant, which is essentially buffered with carbonates and amino acids, so the loading material does not necessarily have to be present.

The method of the present invention preferably comprises washing the loaded anion exchange resin material with a washing buffer in the absence of divalent cations. This cleaning step can be performed by any method known in the art. Wash buffers suitable for cleaning impurities from anion exchange materials without substantially eluting divalent cation-binding proteins are well known in the art. For example, the wash buffer has a pH of at least 1.0 or at least 0.5, in pH units, preferably 5.5-6.5, more preferably 5.9-6.1, below the pH of the loading buffer. Have. The wash buffer contains any salt concentration suitable for washing the anion exchange resin material without eluting the divalent cation binding protein in a significant amount (amount that can be easily determined by those skilled in the art). You may. For example, the wash buffer may contain a suitable buffer of preferably 20 mM Bis-Tris, such as, for example, Bis-Tris, acetate buffer, citrate buffer or phosphate buffer. Preferably, wash 1 and 3 have tris as wash buffer, while wash 2 has MES. Further, for example, EDTA may be contained, preferably 0.5 to 10 mM EDTA, more preferably 1 to 5 mM EDTA, preferably about 2 mM EDTA or the like chelating agent. Further, suitable salts, for example, the following cations and anions of the ^{salt: K +, Na +, Li} +, a salt with _NH ^{4 +,} chlorides, phosphates, sulfates, carbonate, the anion of acetate, etc., washing buffer In order to adjust the conductivity of, for example, in NaCl and the like, it is blended at a concentration of < 200 mM, preferably 100 mM to 200 mM, more preferably 150 mM to 200 mM, further preferably 170 mM to 190 mM, and most preferably 175 mM to 185 mM. May be. In another preferred embodiment of the invention, the wash buffer contains 100-200 mM NaCl. The absolute value of the salt concentration varies depending on the divalent cation-binding protein to be purified, and it is also possible to determine whether the salt concentration needs to be lowered or increased in order for the divalent cation-binding protein to obtain the optimum purity. It is within the recognition of the vendor.

Preferably, according to the method of the invention for purifying a divalent cation bound protein, the second wash step is performed after the first wash step described above. If this cleaning step is a cleaning just before elution, it is carried out with low conductivity. This conductivity is preferably lower than that of the loading step and the first cleaning step. Moreover, more preferably, a third cleaning step can be performed. This embodiment is described in detail above.

Elution of the divalent cation binding protein with an eluent containing the divalent cation can be carried out by a method known in the art. Preferred counterions include Ca ²⁺ , Be ²⁺ , Ba ²⁺ , Mg ²⁺ , Mn ²⁺ , Sr ²⁺ , Zn ²⁺ , Co ²⁺ , Ni ²⁺ and Cu ²⁺ , or a combination thereof. Most preferably, it is calcium. The elution buffer preferably has a pH higher than that of the wash buffer. The pH is preferably at least 0.5, preferably 1.0 higher in pH units. The elution buffer is highly preferably having a pH between 7.5 and 8.5, and even more preferably between 7.9 and 8.1. The elution buffer contains any salt concentration suitable for eluting the divalent cation binding protein from the primary anion exchange resin material in a significant amount that can be easily measured by those skilled in the art without eluting impurities. You may. For example, it is a suitable buffer (eg, HEPES, Tris, preferably 20 mM Tris, Tris / acetate, histidine, Gly-Gly, MOPS, or Tricine, etc.), typically at a concentration in the range of 5-50 mM. It may be contained. It also, in order to control the conductivity of the buffer, a suitable salt, for example, the following cations and ^{anions: K +, Na +, Li} +, and _NH ^{4 +,} chlorides, phosphates, sulfates, carbonates, acetates, etc. It may contain a salt with the anion of (eg, NaCl, etc.) and may be present at a concentration of 150-200 mM. The following is a particularly preferred set of buffers:
Tris: buffer with pH = 8.06 ± 1.0;
HEPES: buffer with pH = 7.7 ± 1.0;
MOPS: buffer with pH = 7.3 ± 1.0;
Tricine: buffer with pH = 8.3 ± 1.0;
Histidine: buffer with pH = 7.6 ± 1.0;
Gly-Gly: buffer with pH = 7.4 ± 1.0;
Bis-tris: buffer with pH = 6.35 ± 1.0;
ACES (N- (2-acetamide) -2-aminoethanesulfonic acid): buffer with pH = 7.0 ± 1.0;
ADA (N- (2-acetamide) -iminodiacetic acid): buffer with pH = 7.0 ± 1.0;
MES: Buffer with pH = about 6.0.

The elution buffer may also contain a suitable salt (eg, NaCl, etc.) to control the conductivity of the wash buffer and may be present at a concentration of 100-200 mM. The absolute value of salt concentration depends on the divalent cation binding protein to be purified, and determining whether a divalent cation binding protein requires a lower or higher salt concentration to obtain optimal purity is possible. It is within the knowledge of those skilled in the art.

After the elution step, the pool of resulting eluate is diluted to reduce conductivity and increase the concentration of divalent cations, preferably calcium. This criterion provides conditions that prevent the product from binding to the second anion exchange resin and promotes binding of the host cell protein. Such dilution is known to those of skill in the art and is carried out by well known methods. For example, by adding one column volume of dilution buffer, Ca increases and conductivity decreases slightly. This diluted solution preferably has a final pH of 7-8, more preferably 7.5-7.9.

Those skilled in the art will appreciate that further operations to impart low conductivity to the resulting pool of eluate are within the definition of diluting the pool of eluate to reduce conductivity. Thus, a further measure that may reduce the conductivity of the pool of eluate is, for example, diluting with a low salt buffer or dialyzing, or changing the composition of the buffer using dialysis filtration.

As used herein, the term "ion exchange resin material" is not based on any particular limitation. According to the present invention, the first and second resins are, for example, agarose-based chromatography materials (eg, Sepharose Fast Flow or Capto), polymer synthetic materials (eg, polystyrene). Suitable for anion exchange chromatography known in the art such as Toyopearls), polystyrene / divinylbenzene (eg, Poros, Source) or cellulose (eg, Cellufine). Both materials are included. In a particular example of the invention, the first and second anion exchange resin materials are sepharose, which is based on modified agarose and its polysaccharide chains are crosslinked to form a three-dimensional network structure. In a preferred embodiment, the first and second anion exchange resin materials are, but are not limited to, resins carrying a primary amine as a ligand (eg, aminohexyl sepharose, benzamidine cepharose, lysine sepharose, or Arginine Sepharose) is included. In another preferred embodiment, the primary and secondary anion exchange resin materials are, but are not limited to, alkylaminoethanes such as diethylaminoethane (DEAE), dimethylaminoethane (DMAE) or trimethylaminoethyl (TMAE). )), Polyethyleneimine (PEI), quaternary aminoalkyl, quaternary aminoethane (QAE), quaternary ammonium (Q), and other resins having positively charged moieties at neutral pH. In a particularly preferred embodiment, the anion exchange resin material is Q-Sepharose Fast Flow (Q-Sepharose FF). According to the method of the present invention, the first and second anion exchange resin materials may be the same or different.

The loading buffer (step d) in the second anion exchange step can be the same as the loading buffer described above in the first anion exchange step. The differences between the two anion exchange steps as provided in accordance with the present invention are essentially as follows:
a) The first anion exchange column is loaded with a starting material, i.e., in a preferred embodiment, a cell culture material. This is a highly impure starting material. In the second anion exchange column, a material that has already been purified to some extent is loaded because it is the material obtained after the elution (and filling) step.
b) The primary anion exchange step is carried out with the divalent cation to elute the product after loading the primary anion exchange column in the absence of the divalent cation.
c) The pH and concentration of the divalent anion of the load for the second anion exchange is, in a preferred embodiment, the pH of the load (d) of the wash after the load (a) (acidic wash, eg wash 2). The pH is selected to be higher than the pH of the load (a) itself if no cleaning step is used. Furthermore, the conductivity of the load (a) is comparable to that of acidic cleaning, but is preferably low if this cleaning is to be performed. The divalent cation concentration, i.e. Ca ⁺⁺ concentration, is higher at load (d) than the pH of the elution buffer in step (b).
d) Thus, in the first anion exchange column, the product binds to the column, but in the second anion exchange column, the product does not.

The divalent cation binding protein of the present invention may be any divalent cation binding protein such as, for example, calcium binding protein and / or vitamin K-dependent protein. In a preferred embodiment, the divalent cation binding protein is selected from the group consisting of Factor II, Factor VII, Factor IX, Factor X, Protein C, Protein S, Anexin and Calmodulin, particularly preferably Factor IX. , Factor VII and Anexin V.

Starting materials (“samples”) for divalent cation-binding proteins may be obtained using methods known to those skilled in the art, such as plasma-derived proteins using CHO cells, proteins produced by gene transfer, etc. Alternatively, it is a protein produced by genetic recombination. Secretory and non-secretory methods for extracting proteins from cell cultures are well known to those of skill in the art. This is to produce recombinant DNA by (i) genetic manipulation, eg, via reverse transcription of RNA and / or amplification of DNA, in order to obtain crude divalent cation-binding proteins. ii) Introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, eg, via electroporation or microinjection, (iii) the transformed cells, eg, in a continuous manner or batch by batch. (Iv) divalent cation-binding protein is expressed, for example, constitutively or by induction, (v) protein is isolated, for example, from a culture group or by harvesting transformed cells. It includes any method known in the art for doing so. Recombinant DNA encoding a divalent cation-binding protein, such as a plasmid, also contains a DNA sequence encoding a selectivity marker for selecting cells that have been successfully transfected with the recombinant DNA. You may.

In a preferred embodiment of the invention, the starting material of the invention is a material containing FIX, preferably plasma containing FIX, or FIX produced by recombinant manipulation. Recombinant production of FIX is well known in the art. The recombinant FIX, rFIX, is secreted into a cell culture supernatant (CCS) according to a preferred embodiment, where the CCS is used as a starting material for the methods of the invention.

The protein may be pre-purified to reduce impurities, for example by gel electrophoresis, chromatography, gel filtration, centrifugation, filtration, precipitation, crystallization, or other methods known in the art. As used herein, the term "impurities" includes any impurities originating from the production of divalent cation binding proteins, such as host cell protein (HCP) impurities, nucleic acid impurities, polypeptide impurities. Includes buffer and salt impurities, impurities originating from cell culture groups, product-related impurities such as dimers or fragments, and combinations thereof.

The methods described herein make it possible to remove or separate, for example, the Inactive and End-Cut Form of FIX from the active FIX product. The method also makes it possible to control or maintain the formation of product-related impurities (eg, degradation products, product agglutinations, particles) at very low levels. Moreover, the method provides conditions for maintaining particularly low levels of activated FIX (FIXa). In addition, the method is particularly effective in separating process-related impurities (CHP HCP, CHO DNA, medium components) from active FIX products.

In particular, an additional impurity found in FIX preparations, as well as FIX pharmaceutical preparations, is activated FIX, i.e. FIXa. FIXa has been shown to adversely affect the resulting FIX preparation as it causes thrombus formation. Thus, it is highly desirable to provide a method of reducing the content of FIXa in the FIX preparation.

The method of the present invention achieves this object by preventing the formation of FIXa in the preparation obtained by the method.

In an advantageous and surprising manner, if the starting material used is FIX produced by recombinant techniques in cell culture or cell culture supernatant, the content of host cell protein (HCP), especially CHO cell HCP. It is also possible to reduce.

In a further advantageous and surprising way, it is also possible to reduce the content of host cell DNA, especially CHODNA, in the composition obtained by the method of the invention.

According to the above, further improvements are provided in the yield and specific activity provided by the present invention.

In a preferred embodiment, the divalent cation-binding protein purified according to the methods of the invention is at least 95% w / w, more preferably at least 98% w / w, for all proteins with respect to host cell protein (HCP) impurities. It has a purity of w, more preferably at least 99% w / w, most preferably at least 99.5% w / w, of the divalent cation-binding protein. Therefore, in a preferred embodiment, the content of HCP impurities in the purified divalent cation-binding protein is less than 5% w / w, more preferably less than 2% w / w, more preferably less than 1% w / w. Most preferably, it is less than 0.5% w / w. The proportion of HCP impurities refers to the product, i.e. the w / w of the purified divalent cation binding protein, which can be measured, for example, by HPLC or ELISA.

Furthermore, in another aspect of the invention, there is provided a purified divalent cation binding protein that can be obtained or obtained by the methods of the invention. Kits are also provided that include means for carrying out the methods of the invention. In particular, the kits of the invention are of loading buffers and / or eluents and / or wash buffers, and / or divalent cations that are suitable for purifying divalent cation binding proteins using anion exchange resin materials in accordance with the invention. It may contain a solution. In a preferred embodiment, the loading buffer, wash buffer and / or eluent are as described above. Furthermore, the kit of the present invention may contain a suitable anion exchange resin material.

The invention further relates to the use of the methods of the invention described above and / or the kits of the invention described above for purifying divalent cation binding proteins.

The present invention provides a method for efficiently purifying a divalent cation-binding protein, which can produce a product in a high yield and at the same time significantly reduce protein process-related impurities by using an anion exchange resin material. To do.

The pool of eluate diluted in the primary anion exchange purification step is then loaded into a secondary anion exchange column that does not bind under conditions where the divalent cation binding protein is applied. As a result, the divalent cation-binding protein contained in the column effluent of the second anion exchange purification has high purity and high specific activity. Furthermore, it has a low content of (CHO) HCP, a low content of (CHO) DNA and a low content of FIXa.

The content of FIXa is measured by its activity, which is expressed as% activity against Factor IX activity.

The efficacy of recombinant FIX (rFIX) products (in the international unit, IU) is based on the well-known and recognized in vitro one-step coagulation assay for factor IX concentrates from the World Health Organization (World Health Organization). Determined using a reference adjusted to WHO) international standards. One international unit is the amount of FIX activity present in pooled human normal plasma (1 mL).

FIXa in the product is measured using an assay commonly known to those of skill in the art. As an example, a commercially available color-developing FIXa kit (Rox FIX-A, etc., product number 950030; Rossix, using a reference adjusted to the World Health Organization (WHO) international standards for Factor IX. Molndal, Sweden). To carry out such an assay, human FX is activated by FIXa in the presence of FVIII, thrombin, calcium and phospholipids to FXa. The amount of FXa produced is measured with a specific FXa substrate that releases p-nitroaniline in an amount proportional to the amount of FXa upon cleavage. The assay is highly sensitive to FIXa with a lower limit of 0.10 mIU / mL.

The levels of pre-activated FIX (rFIXa) in the final product (the product obtained by the method of the invention) were much lower. The permissible limit is set as < 0.10% FIXa (color-developing IU / mL) / FIX activity (coagulant IU / mL). The actual FIXa content of the 15 test batches of the present invention, however, was as low as < 0.02% FIXa / FIX. All FIX preparations obtained according to the purification process of the present invention had values below 0.02% FIXa / FIX. One BeneFIX lot (E94791 as a comparative product available on the market) was analyzed in the same assay and the relative content of FIXa was measured to be 0.11%.

Taken together, it was clear that the FIXa content of all lots was consistently low, up to 10-fold lower than the content of comparable products.

In particular, the method of the present invention is based on the following principle. In general, the bond between the protein and the anion exchange resin material becomes stronger at low conductivity and high pH values. Conversely, the bond between the protein and the anion exchange resin material is weakened at high conductivity and low pH values. In the method of the invention, the divalent cation binding protein is preferably loaded and / or washed at a low pH, as described in detail above, which further exchangs the first anion of the divalent cation binding protein. Allows binding to materials and does not impair the structural integrity or activity of divalent cation binding proteins. Many impurities in the protein do not bind to the first anion exchange resin material, thus the binding of impurities to the first anion exchange resin material is greatly reduced, while the product does indeed bind to the first anion exchange column. Under these conditions, protein impurities that bind to the primary anion exchange resin material and are not washed off are prevented from being eluted together by raising the pH during elution. Increasing the pH of the eluent causes all proteins to bind more strongly to the primary anion exchange resin material, but the divalent cations interact specifically with the elution-inducing product. The eluate of the first anion exchange column adjusts the conductivity (as described above, equal to or lower than the acidic wash) and pH (more than the load (a) or acidic wash). It is conditioned to raise the pH to at least 0.5 higher and to raise the concentration of divalent cations (higher than the concentration at load (a)). These criteria provide conditions for selective binding of impurities to anion exchange resins and selective non-bonding of products. According to the method of the present invention, only the divalent cation binding protein does not bind to the second anion exchange material due to the divalent cation present in the eluate. In contrast, raising the pH during elution in step (b) is very specific for the anion exchange elution operation, and raising the pH for loading in step (d) is a negative chromatograph compared to acidic washing. It is very special in imaging, i.e. anion exchange chromatography (as mentioned above, the purified protein is considered to flow through because the protein generally binds more strongly to the anion exchange resin material at high pH values). By using the eluate from the primary anion exchange resin material with at least one divalent cation, the method of the invention is surprisingly and advantageously the product of a divalent cation binding protein by anion exchange chromatography. Achieve excellent purification.

In particular, increasing the pH during elution of the Ca-induced product in step (b) (the elution buffer is higher in pH than the loading buffer in step (a) or the acidic wash in step (a)) and Adjusting the load for step (d) (higher pH than acid washing in step (a) and higher divalent cation than eluent in step (b)) is a divalent (Ca) bond. It provides selective purification of the protein and prevents impurities from eluting together. Loading of a secondary anion exchange resin material at high pH and high calcium (as an example of a divalent cation) purifies by binding impurities to the secondary anion exchange resin material while supplementing with the divalent cation. Inhibits the binding of power proteins. According to the present invention, the above conditions result in high purity and high yield of divalent cation binding protein. In the method of the present invention, for example, a solution of a protein is loaded onto an anion exchange resin material at low pH, the product is eluted at high pH with an eluent at least one divalent cation, and the eluate is high. Provided is a method of significantly reducing impurities in process-related polypeptides by loading on a secondary anion exchange resin material at pH and under conditions of high concentration of divalent cations and collecting flow-throughs.

Various modifications and variations of the methods and products described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. The present invention is described in connection with certain preferred embodiments, but should not be unduly limited to such embodiments.

The purification method is based on the unique biochemical properties of vitamin K-dependent proteins. For example, in the Gla domain, about 12 negative charges are focused within the short amino acid stretch. This negative charge can be neutralized or converted to a positive charge by adding a divalent cation (eg, Ca ²⁺ ) to the system. Based on this action, rFIX can be eluted from the gel by binding to an ion exchange resin in one particular charge state and converting the charge by removing (or adding) Ca ²⁺ . The principle of this separation is called "pseudo-affinity chromatography". This principle is also reflected in those vitamin K-dependent proteins that do not contain the Gla-domain. For example, the method of the invention applied to the purification of divalent cation binding proteins such as rFIX introduces a pH shift into its loading, washing and / or elution buffer to introduce the product and inactive FIX species and CHO host cells. Significantly improves the removal of process-related impurities such as proteins.

Purification procedure of rFIX The present inventors have developed a recombinant CHO cell line expressing recombinant human Factor IX based on the CHO DXB11 parenteral cell line. The cell line was genetically engineered to co-express recombinant human furin and ameliorate the intracellular mutation of proFIX to FIX. In the production process, cells were cultured in suspension in chemostat mode and adapted to synthetic medium without the addition of mammalian or plasma-derived proteins. Soybean peptone was added to the medium to improve clone production.

In the chemostat production campaign, the cultured harvest of the collected cells is subjected to deep filtration with a Cuno depth filter (filter with positive zeta potential) and 0.2 pm membrane filtration with a PVDF or PES filter membrane. Purify and remove cells and cell debris. The filtered cell-free harvest is used as a starting material for developing the purification process of rFIX.

The large-scale purification process applied to the clinical production of rFIX begins with a capture step by anion exchange chromatography at Q-Sepharose Fast Flow, where the purified harvest is supplemented with 2 mM EDTA (see Table 1). , Load at a pH of about 7.0-7.4. After loading (the loading buffer was a cell-free culture supernatant of a CHO cell line expressing rFIX) and washing 1, washing 2 at low pH was performed at pH 6.0, and inactive FIX. Remove seeds and bound CHO proteins. Perform a third wash with low conductivity and pH 7.3-7.5 to prepare a column for elution. From the column, the bound FIX is eluted with a buffer containing calcium and having a pH (8.0) significantly higher than the pH of wash 2. This procedure results in selective elution of FIX and very low co-eluting of product and process related impurities (inactive FIX species and CHO host cell proteins). About 99.5% of the CHO HCP protein added to the column was removed in this step (CHO HCP reduction factor was about 190) and the specific activity of FIX increased about 1.5-fold. Table 2 below summarizes the composition of the buffer for the Q-Sepharose Capture step (first anion exchange step).

Virus inactivation of solvent / cleaning agent is performed by adding 1% Triton X-100 / tri-n-butyl phosphate and 0.3% polysorbate 80 to the pool of eluate in the first anion exchange purification step. .. The S / D treated FIX solution is then diluted with a calcium-containing buffer to adjust the calcium concentration to about 6 mM and reduce the conductivity to 15-18 mS / cm (RT). The pH of the solution dropped slightly from 8.0 to about 7.6-7.8.

The S / D-treated and diluted FIX solution, which does not bind to FIX, is then applied to the first refining step at Q-Sepharose Fast Flow under applicable conditions (see Table 3 below). The remaining CHO host cell protein (CHO HSP) has a higher pH than Wash 2 (capture step with Q-Sepharose Fast Flow) and lower conductivity than the pool of eluate in the capture step, resulting in anion exchange. Can bind to resin.

The results obtained with this improved purification method are summarized in Tables 4 and 5 below; Tables 4 and 5 show the results of the two subsequent experiments.

Claims (8)

A pharmaceutical composition comprising a recombinant FIX produced in CHO cells, comprising less than 10 pg / ml of CHO DNA and 0.02% or less of FIXa (activated FIX) / FIX, wherein the recombinant FIX is A pharmaceutical composition having a specific activity of 270 to 320 I.U./mg FIX. The pharmaceutical composition according to claim 1, wherein the recombinant FIX has a specific activity of 280 to 300 I.U./mg FIX. The pharmaceutical composition according to claim 1, which comprises CHO DNA of less than 5 pg / ml. The pharmaceutical composition according to claim 1, which comprises CHO DNA of less than 1 pg / ml. The pharmaceutical composition according to claim 1, which comprises FIXa (activated FIX) activity / unit of FIX coagulation activity of less than 1 mU. The pharmaceutical composition according to claim 1, which comprises FIXa activity / unit of FIX coagulation activity of less than 0.75 mU. The pharmaceutical composition according to claim 1, which comprises FIXa activity / unit of FIX coagulation activity of less than 0.5 mU. The pharmaceutical composition according to claim 1, which comprises FIXa activity / unit of FIX coagulation activity of less than 0.3 mU.